Improvements are provided for in vitro culturing of a human cancer cell of the lymphocyte lineage (e.g., leukemia or lymphoma), another lymphoblast, or a precursor thereof, especially T-cell acute lymphoblastic leukemialymphoma (T-ALL).
T-ALL is an aggressive malignancy for which optimization of chemotherapy has led to steady improvements in outcome for pediatric patients. But 20-25% of pediatric patients fail current chemotherapy regimens, and outcomes in adult patients are not as good as for children. For this group of patients, new effective therapies are desperately needed. In addition, current effective therapies in children require up to two years of treatment with highly toxic drug combinations. Therefore, there is a need for more effective and less toxic treatments in this disease.
Efficient testing and validation of novel therapies would be greatly enhanced by development of a robust in vitro culture model that allows direct assay of primary human T-ALL blasts. Most efforts to date have relied heavily upon established cell lines, which are adapted to growth in high concentrations of serum and have certain molecular features (e.g., high frequency of p53 mutations) that are not present in primary tumors. While primary patient T-ALL samples can be expanded as xenografts in immunodeficient mice (see Armstrong et al., Blood 113:1730-1740, 2009; Chiu et al., Blood 116:5268-5279, 2010; Cox et al., Blood 109:674-682, 2007; Medyouf et al., Blood 115:1175-1184, 2010), such in vivo studies are costly and time consuming. They are often complicated by the poor health of immunodeficient mice, precluding optimal drug dosing.
Several groups have reported cell culture of primary human T-ALL in vitro with supplemental cytokines, although results were typically highly variable and, in many cases, cultures have likely undergone crisis prior to the expansion phase, suggesting the outgrowth of a minor subpopulation or variant subclone. A co-culture system for human T-ALL uses a feeder layer (i.e., mouse stromal cells) that express Notch ligand Delta-like-1 (DL1) to activate Notch signaling and further sustain blast cell growth (Armstrong et al., Blood 113:1730-1740, 2009; Chiu et al., Blood 116: 5268-5279, 2010). For example, Pflumio medium containing a mixture of fetal calf serum and human serum was used to culture human T-ALL in vitro over an MS5-DL1 feeder layer. But there is substantial variability in different serum lots for their ability to support cell maintenance and propagation.
Thus, an in vitro culturing system for primary T-ALL to study specific gene mutation, chromosome rearrangement, epigenetic changes in DNA methylation or chromatin, drug resistance, expression of cell markers and their function, immunogenicity, progression through the cell cycle and apoptosis, sensitivity to a therapeutic agent, or any combination thereof would be desirable. In vitro culturing may be used to identify a precursor cell that gives rise to T-ALL (i.e., cancer stem cell), to differentiate T-ALL, or to determine whether T-ALL disease is clonal. For heterogeneous cells, a T-ALL subpopulation may be subcloned by cell separation or dilution.
Here, a serum-free, chemically-defined medium previously used for in vitro culture of mammary epithelium (Ince et al., Cancer Cell 12:160-170, 2007) is modified by excluding cholera toxin and including additional components to support the growth of T-ALL. US20080299540 described a similar cell culture medium, different from the medium disclosed herein, without mentioning supporting the maintenance and propagation of T-ALL.
It is an objective to provide an in vitro culture system for improved maintenance and propagation of human cancer cells of the lymphocyte lineage (e.g., leukemia or lymphoma), other lymphoblasts, or precursors thereof, especially T-cell acute lymphoblastic leukemialymphoma (T-ALL). The system may be used to expand or to examine the cell. A chemically-defined, cell culture medium and a method for in vitro culturing of one or more human cell(s) are provided as improvements over the prior art.